Worm Breeder's Gazette 12(5): 20 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Transposon Induced Deletion Mutagenesis, A General Method

Annegien Broeks, Richard Zwaal, Ronald H.A. Plasterk

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Netherlands Cancer Institute, Plesmanlaan 121,1066 CX Amsterdam
phone +31 20 5122081, fax +31 20 6172625.

To investigate the function of a gene one can analyze the phenotype of a null allele of that gene. Methods have been described to obtain Tc1 insertion alleles of genes (PCR and sib selection, A.M. Rushforth et al. WBG 11(5):65; mutant bank, R.H.A. Plasterk et al. WBG 12(4):11). Tc1 insertions are most frequently found in intron sequences. In some cases all or part of Tc1 can also be spliced out from exons, with the possibility that the function is not completely lost (A.M. Rushforth et al. in press). Therefore neither Tc1 intron nor exon insertions can be considered guaranteed null alleles.

Some revertants of unc-22 ::T c1 alleles were found to have lost Tc1 plus 1-2 kb flanking sequences (J.E. Kiff et al. Nature 331:361-363 '8o. These deletions probably occur during repair of DNA breaks caused by Tc1 excision. Based on this observation we used Tc1 insertions as a starting point to isolate deletion mutants. 100 Cultures are started with 10 animals each; the DNA is analyzed using PCR. The primers for PCR are chosen a) to select for unidirectional deletions, or b) to select for bidirectional deletions. The distance between the primers is chosen 4.6 kb (3 kb genomic sequences + 1.6 kb Tc1 sequences), thus no product is found without a flanking deletion. A 3 kb somatic excision product can be found, but it is outcompeted in the PCR if a deletion allele is present.

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Deletions occurred with a frequency of 10 2 to 10-3 per allele per generation. Both unidirectional and bidirectional deletions have been found, the size varying from 900 bp to 2 kb. Thus far we have obtained 3 clonal deletion mutants (1 for pgp-1 and 2 for gpa-1 ) and we are in the course of cloning several other deletion derivates that have been detected ( pgp-1 , pgp-3 , ceh-13 and gpa-2 ).

More Tc1 insertions will be studied to investigate whether the unexpected high frequency of deletion alleles generated from Tc1 alleles is a general phenomenon. Since we have observed deletion derivates in all genes we looked at, it seems to be a general method. This method allows the use of all exon and intron insertions isolated from the mutant bank for the isolation of null alleles.

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